Galleria mellonella as model host for the trans-kingdom pathogen Fusarium oxysporum

Fusarium oxysporum, the causal agent of vascular wilt disease, affects a wide range of plant species and can produce disseminated infections in humans. F. oxysporum f. sp. lycopersici isolate FGSC 9935 causes disease both on tomato plants and immunodepressed mice, making it an ideal model for the comparative analysis of fungal virulence on plant and animal hosts. Here we tested the ability of FGSC 9935 to cause disease in the greater wax moth Galleria mellonella, an invertebrate model host that is widely used for the study of microbial human pathogens. Injection of living but not of heat-killed microconidia into the hemocoel of G. mellonella larvae resulted in dose-dependent killing both at 30 °C and at 37 °C. Fluorescence microscopy of larvae inoculated with a F. oxysporum transformant expressing GFP revealed hyphal proliferation within the hemocoel, interaction with G. mellonella hemocytes, and colonization of the killed insects by the fungus. Fungal gene knockout mutants previously tested in the tomato and immunodepressed mouse systems displayed a good correlation in virulence between the Galleria and the mouse model. Thus, Galleria represents a useful non-vertebrate infection model for studying virulence mechanisms of F. oxysporum on animal hosts.     

Galleria mellonella infected with Bacillus thuringiensis involves Hsp90

Insects are good models for studying the innate immune response. We report that Galleria mellonella larvae infected with entomopathogenic bacteria Bacillus thuringiensis kurstaki show changes in the level of Hsp90. Our experimental approach was to pre-treat larvae with the Hsp90-binding compound, 17-DMAG, before infection with B. thuringiensis. We show that pre-treated animals display a higher level of immune response. This was mainly manifested by enhanced action of their hemolymph directed toward living bacteria as well as lysozyme activity digesting bacterial peptidoglycan. The observed phenomenon was due to the higher activity of antimicrobial peptides which, in contrast to healthy animals, was detected in the hemolymph of the immunestimulated larvae. Finally, the physiological significance of our observation was highlighted by the fact that G. mellonella pre-treated with 17-DMAG showed a prolonged survival rate after infection with B. thuringiensis than the control animals. Our report points to a role for Hsp90 in the immune response of G. mellonella after infection with B. thuringiensis at the optimal growth temperature.     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.     

Microconidia germination of the tomato pathogen Fusarium oxysporum in the presence of root exudates

Abstract

In this study we assessed microconidia germination of the tomato pathogens F. oxysporum f. sp. lycopersici (Fol) and F. oxysporum f. sp. radicis-lycopersici (Forl) in the presence of root exudates. Tomato root exudates stimulated microconidia germination and the level of stimulation was affected by plant age. Treatment of root exudates with insoluble polyvinylpolypyrrolidone, which binds phenolic compounds, indicated that tomato root exudates contain phenolic compounds inhibitory to F. oxysporum microconidia germination. Our study indicates that tomato root exudates similarly stimulate microconidia germination of both Fol and Forl. However, individual F. oxysporum strains differ in the degree of germination response to the root exudates. Furthermore, root exudates from non-host plants also contain compounds that stimulate microconidia germination of Fol. In general, the effects of root exudates from non-host plants did not differ considerably from those of tomato. The ability of phenolic compounds to inhibit germination of Fol seems not to be plant-specific.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

Detection of serpins involved in cellular immune response of Rhipicephalus microplus challenged with fungi

The understanding of tick physiology and immune system is important to improve the effective control of this ectoparasite. Invertebrates' innate immune response is activated when the organism is challenged with pathogens. The present study describes the changes of serine proteinase inhibitors (serpins) and in the number of circulating haemocytes involved in cellular immune defence of Rhipicephalus microplus engorged females challenged with the entomopathogenic fungi Metarhizium anisopliae or Beauveria bassiana, or with the non-entomopathogenic fungus Fusarium oxysporum. The cell-free haemolymph was separated from haemocytes by centrifugation and cells were re-suspended in phosphate buffer pH 7.2. The proteins of haemocytes were analysed by SDS-PAGE and the segments of the 1D gel were submitted to protein digestion with trypsin. The peptides were analysed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS). The analysis by mass spectrometry allowed the identification of several proteins through the search in the database built based on public banks of Ixodidae and Argasidae. In haemocytes, many proteins were identified highlighting serpins. The results showed that the entomopathogenic fungi M. anisopliae or B. bassiana reduced the amount of serpins, while F. oxysporum increased. The present study reports, for the first time, the variation of serpins in haemocytes of R. microplus engorged females infected by fungi.     

iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.Stable isotope labeling techniques have become very popular in recent years to perform quantitative mass spectrometry experiments with high precision and accuracy. In contrast to label-free approaches, multiplexed isotopically labeled samples can be simultaneously analyzed resulting in increased reproducibility and accuracy for quantification of peptides and proteins from different biological states. Isotopic labeling strategies can be grouped into two major categories: isobaric labels and nonisobaric labels. In the former category are iTRAQ¹ (isobaric tags for relative and absolute quantification (1)) and TMT (tandem mass tags (2)) mass tags. In the nonisobaric labeling category are methods such as mTRAQ (mass differential tags for relative and absolute quantification), stable isotope labeling by amino acids in cell culture (SILAC (3)), and reductive dimethylation (4). Isobaric labeling techniques allow relative quantification of peptides based on ratios of low m/z reporter ions produced by fragmentation of the precursor ion, whereas nonisobaric labeling yields precursors with different masses that can be directly quantified from MS1 intensity. iTRAQ and mTRAQ reagents provide a great opportunity to directly compare capabilities of reporter and precursor ion quantification since both labels have identical chemical structures and differ only in their composition and number of ¹³C, ¹⁵N, and ¹⁸O atoms. In fact, iTRAQ-117 and mTRAQ-Δ4 are identical mass tags with a total mass of 145 Da (Fig. 1A). To achieve 4-plex quantification capabilities for iTRAQ labels, the composition of stable isotopes is arranged in a way to obtain the reporter ion/balancing group pairs 114/31, 115/30, 116/29, and 117/28 (1). Three nonisobaric mTRAQ labels were generated by adding or removing four neutrons to the mTRAQ-Δ4 label resulting in mTRAQ-Δ8 and mTRAQ-Δ0, respectively. Both iTRAQ and mTRAQ reagents are available as N-hydroxy-succinimide esters to facilitate primary amine labeling of peptides.Open in a separate windowFig. 1.A, Labeling strategy for comparative evaluation of iTRAQ and mTRAQ tags. Peptides were labeled with the indicated iTRAQ and mTRAQ reagents for combined phosphoproteome and proteome analysis. B, Selection of optimal instrument methods for analysis of iTRAQ- and mTRAQ-labeled peptides. Unfractionated proteome samples (1 ug) and phosphoproteome samples (enriched from 250 μg peptides) were analyzed for iTRAQ samples with a CID/HCD-Top8 method, whereas for mTRAQ we compared CID-Top16 acquisition to HCD-Top8. Note that duty cycle times were for all instrument methods ∼3.1 s.One potential advantage of an iTRAQ labeling strategy is its additive effect on precursor intensities when samples are multiplexed, resulting in increased sensitivity. However, iTRAQ ratios have been demonstrated to be prone to compression. This occurs when other nonregulated background peptides are co-isolated and cofragmented in the same isolation window of the peptide of interest and contribute fractional intensity to the reporter ions in MS2-scans (5–7). Because most peptides in an experiment are present at 1:1:1:1 ratios between multiplexed samples, all ratios in the experiment tend to be dampened toward unity when cofragmentation occurs. This inaccuracy led to the development of mTRAQ labels to facilitate accurate precursor-based quantification of proteins initially identified in iTRAQ discovery experiments with targeted assays, such as multiple reaction monitoring (MRM) (8). Although iTRAQ has been widely used in discovery-based proteomics studies, mTRAQ has only appeared in a small number of studies thus far (8).In this study we investigated the advantages and disadvantages of iTRAQ and mTRAQ labeling for proteome-wide analysis of protein phosphorylation and expression changes. We selected epidermal growth factor (EGF)-stimulated HeLa cells as a model system for our comparative evaluation of iTRAQ and mTRAQ labeling, as both changes in the phosphoproteome (9) as well as the proteome (10) are well described for EGF stimulation. We show that iTRAQ labeling yields superior results to mTRAQ in terms of numbers of quantified phosphopeptides, proteins and regulated components. By means of spike-in experiments with GluC generated peptides of known ratios we find that iTRAQ quantification is more precise but less accurate than mTRAQ due to ratio compression. We identify a linear relationship of observed versus expected logarithmic GluC generated peptide ratios as well as for logarithmic iTRAQ and mTRAQ ratios of the phosphoproteome and proteome analysis. This indicates a uniform degree of ratio compression over two orders of magnitude throughout iTRAQ data sets and explains why iTRAQ ratio compression does not compromise the ability to detect regulated elements in these experiments.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

Fusarium oxysporum is one of the most abundant and diverse fungal species found in soils and includes nonpathogenic, endophytic, and pathogenic strains affecting a broad range of plant and animal hosts. Conidiation is the major mode of reproduction in many filamentous fungi, but the regulation of this process is largely unknown. Lysine acetylation (Kac) is an evolutionarily conserved and widespread posttranslational modification implicated in regulation of multiple metabolic processes. A total of 62 upregulated and 49 downregulated Kac proteins were identified in sporulating mycelia versus nonsporulating mycelia of F. oxysporum. Diverse cellular proteins, including glycolytic enzymes, ribosomal proteins, and endoplasmic reticulum–resident molecular chaperones, were differentially acetylated in the sporulation process. Altered Kac levels of three endoplasmic reticulum–resident molecular chaperones, PDI^K70, HSP70^K604, and HSP40^K32 were identified that with important roles in F. oxysporum conidiation. Specifically, K70 acetylation (K70ac) was found to be crucial for maintaining stability and activity of protein disulphide isomerase and the K604ac of HSP70 and K32ac of HSP40 suppressed the detoxification ability of these heat shock proteins, resulting in higher levels of protein aggregation. During conidial formation, an increased level of PDI^K70ac and decreased levels of HSP70^K604ac and HSP40^K32ac contributed to the proper processing of unfolded proteins and eliminated protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation in F. oxysporum.     

Food quality affects the expression of antimicrobial peptide genes upon simulated parasite attack in the larvae of greater wax moth

Predator‐prey interactions are an important evolutionary force affecting the immunity of the prey. Parasitoids and mites pierce the cuticle of their prey, which respond by activating their immune system against predatory attacks. Immunity is a costly function for the organism, as it often competes with other life‐history traits for limited nutrients. We tested whether the expression of antimicrobial peptides (AMP) of the larvae of the greater wax moth Galleria mellonella (L.) (Lepidoptera: Pyralidae) changes as a consequence of insertion of a nylon monofilament, which acts like a synthetic parasite. The treatment was done for larvae grown on a high‐quality vs. a low‐quality diet. The expression of Gloverin and 6‐tox were upregulated in response to the insertion of the nylon monofilament. The expression of 6‐tox, Cecropin‐D, and Gallerimycin were significantly higher in the ‘low‐quality diet’ group than in the ‘high‐quality diet’ group. As food quality seems to affect AMP gene expression in G. mellonella larvae, it should always be controlled for in studies on bacterial and fungal infections in G. mellonella.     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.